Effects of a hydropower project on a high‐value Asian elephant population

Abstract Habitat loss and fragmentation are leading contributors to the endangered status of species. In 2006, the Nakai Plateau contained the largest known Asian elephant (Elephas maximus) population in the Lao People's Democratic Republic (Lao PDR), and the population was among those with the highest genetic diversity reported for Asian elephants. In 2008, completion of the Nam Theun 2 hydroelectric dam inundated much of the Plateau, resulting in the loss of 40% of elephant habitat. We studied elephant presence, movements, and the incidence of human–elephant conflict (HEC) on the Nakai Plateau and surrounding areas from 2004 to 2020, before and for 12 years after dam completion. To examine contemporary population dynamics in the Nakai elephants, we used genetic sampling to compare minimum population numbers, demography, and levels of genetic diversity from the wet and dry seasons in 2018/2019, 10 years after dam completion, with those reported in a pre‐dam‐completion genetic survey. After dam completion, we found a major increase in HEC locally and the creation of new, serious, and persistent HEC problems as far as 100 km away. While we were unable to compare estimated population sizes before and after dam completion, our data revealed a decrease in genetic diversity, a male‐biased sex ratio, and evidence of dispersal from the Plateau by breeding‐age females. Our results raise concerns about the long‐term viability of this important population as well as that of other species in this region. Given that hydropower projects are of economic importance throughout Laos and elsewhere in southeast Asia, this study has important implications for understanding and mitigating their impact.


| INTRODUC TI ON
Development activities such as roads, dams, and other infrastructure projects can result in the loss and fragmentation of wildlife habitat, block migration routes, and facilitate poaching of wild animals, including elephants (Choudhury, 2004). The Asian elephant (Elephas maximus) is listed as Endangered in the IUCN Red List of Threatened Species (Williams et al., 2020) and is included in CITES appendix I. It is threatened by habitat loss/transformation and fragmentation, poaching, and removals from the wild, both legal and illegal (Leimgruber et al., 2003;McWilliam et al., 2010;Sukumar, 1989). As habitat becomes less suitable due to decreased area and/or increased fragmentation, it becomes less able to support a viable population over the long term (Leimgruber et al., 2003). In addition, although elephants and other species may remain in an area following habitat transformation, doing so can place them and nearby human populations at extreme risk of conflict (Kushwaha & Hazarika, 2004).
In areas where human and elephant populations overlap, crop raiding by elephants is the main source of conflict (Cabral de Mel et al., 2022;IUCN, 2023). Elephants trample and feed in cultivated fields, particularly at night, resulting in losses of crops and damage to structures that store harvested grains, losses that are particularly acute for low-income subsistence farmers whose fields are near forests and protected areas. In addition to severe economic losses, human-elephant conflict (HEC) results in an alarming number of deaths of both elephants and humans. For instance, between 2015 and 2018 an average of 124 elephants and 571 humans were killed annually in India (Cabral de Mel et al., 2022). Despite efforts by local governments and non-governmental organizations to prevent and mitigate the effects of HEC (Shaffer et al., 2019), it breeds hostility from local populations and erodes support for elephant conservation.
The Lao People's Democratic Republic (Lao PDR, hereafter referred to as Laos) historically contained extensive elephant habitat and travel corridors (Khounboline, 2011). While elephant populations have declined, as they have across much of southeast Asia, central Laos, and especially the Nakai Plateau area, was thought to contain one of the two most important elephant populations in the country (Duckworth & Hedges, 1998). More generally, the Nakai-Nam Theun National Park (NNT NP), spanning the north-east half of the Nakai Plateau into the Annamite Mountains up to the Lao-Vietnam border, is considered a biodiversity hotspot (Myers et al., 2000).
Over recent decades, the countries of Southeast Asia have experienced rapid economic and population growth, leading to increases in energy needs (Sakti et al., 2023).
Between 2005 and 2008, the Nam Theun 2 (NT2) hydroelectric dam, one of the largest dam projects in Southeast Asia, was constructed on the Nam Theun River. To assess the conservation significance of the elephant population on the Nakai Plateau and inform wildlife management strategies to help mitigate the impact of the NT2 project prior to the creation of the reservoir on the plateau, a dung-count-based survey and a simultaneous genetic capturemark-recapture (CMR) study were conducted from February to May 2006 (Ahlering, Hedges, et al., 2011;Hedges et al., 2013). These studies estimated the Nakai population at 141 individuals (95% CI = [95,208]) using the dung-count method and 132 (95% CI = [120,149]) using the genetic CMR method, with 102 unique genotypes identified (Ahlering, Hedges, et al., 2011). The studies found that the Nakai population had a combination of high genetic diversity, largely intact social structure, and relatively large size, all of which identified it as having high conservation value (Ahlering, Hedges, et al., 2011;Hedges et al., 2013). In subsequent comparisons of the results of the 2006 Nakai study with elephant populations elsewhere in Asia, Ahlering et al. (2020) reiterated the importance of the high levels of genetic diversity found in the Nakai elephant population.
The Convention on Biological Diversity (UNEP, 1992) defined genetic diversity as one of the three pillars of biodiversity (Hoban et al., 2021) as it provides the raw material on which natural selection acts. In the early stages of population reduction and fragmentation, diversity in the form of rare alleles may be lost and with them the heritable variation needed to adapt to environmental changes, including changes in climate and the emergence of novel pathogens (Wernberg et al., 2018). As population numbers decline, alleles continue to be lost through random genetic drift. If this decline is accompanied by barriers to gene flow, inbreeding can further erode the ability of a population to maintain viability over the long term (Frankham, 2022).
Thus, monitoring the levels of genetic diversity is an essential component of population management (Hoban et al., 2021).
The Nakai Plateau underwent a major habitat transformation following the completion of the NT2 hydroelectric dam in April 2008. Prior to dam completion, most of what became the 450 km 2 Nam Theun reservoir was forested, and an estimated 40% of suitable elephant habitat on the plateau was lost as a result (McWilliam et al., 2010). Here, we combine the results of studies of elephant presence, movements, and the incidence of HEC in the Nakai Plateau and surrounding areas before and for 12 years after dam completion with a genetic study of the Nakai elephant population 10 years after dam completion (Eggert & Ruiz-Lopez, 2012). Our objectives were to (1) assess the geographic patterns of elephant presence and dispersal, mainly relating to human-elephant conflict (HEC), before and after the completion of the NT2 dam, (2) compare population size, demography, and levels of genetic diversity in the Nakai elephants before (Ahlering, Hedges, et al., 2011;Hedges et al., 2013) and 10 years after the completion of the dam, and (3) assess differences between the wet and dry seasons in the 2018/2019 Nakai elephant population to more fully understand contemporary movement patterns and population dynamics in the Nakai elephants.
2 | ME THODS 2.1 | Geographic patterns of elephant presence and human-elephant conflict Starting in October, 2004, prior to dam construction, several studies were conducted to better understand the dynamics of elephant populations and the incidence of HEC in the Nakai Plateau and surrounding areas (Hedges et al., 2007;McWilliam et al., 2010;Tyson & Phakphothong, 2015;Tyson & Rasphone, 2013;Tyson & Stremme, 2020). From October, 2004, to August, 2020, the Wildlife Conservation Society (WCS), in collaboration with the District Agriculture and Forestry Offices (DAFO) of affected districts, used monitoring teams to visit villages both on and off the Nakai Plateau ( Figure 1) at least once a month in response to HEC reports.
Beginning in 2009, a team from the Nam Theun 2 Power Company (NTPC) partnered with WCS and DAFO in these efforts. At each visit, monitoring teams recorded the GPS location of each HEC incident, any available details about the elephants involved such as sex, age and group size, and the types of damage incurred.
Between January and March 2011, a study focusing on the Sepon Mine area (Figure 1), some 100 km from the Nakai Plateau, was conducted in response to reports of increases in elephant presence and HEC (Hedges & Hallam, 2011). In addition to monitoring of HEC, teams collected dung samples using a Capture-Mark-Recapture (CMR) protocol (Hedges & Lawson, 2006) for a separate study designed to estimate population size and demography (Eggert & Ruiz-Lopez, 2012).
In 2009, a group of three elephants moved away from the Nakai Plateau into the Gnommalath Plain area and regions southwest of the plateau, subsequently causing fear among villagers, crop-raiding, and property damage in villages that had no previous history of HEC.
The "group of three" (G3) elephants were routinely aggressive toward villagers, even during daylight (Tyson & Phakphothong, 2015;Tyson & Rasphone, 2013;Tyson & Stremme, 2020). By 2019, the group had grown to 6 individuals. Monitoring of movements and reports of HEC by these individuals was conducted by WCS/DAFO/ NTPC teams as in the other regions near the Nakai Plateau.

| Sampling for genetic analyses
To characterize the Nakai Plateau elephant population 10 years after dam completion, we collected fresh dung samples on the Nakai Fresh dung piles were identified using the criteria of Hedges and Lawson (2006). From each fresh dung pile, approximately 10 g of feces were preserved in 40 mL polypropylene tubes in Queen's College Buffer (QCB, Amos et al., 1992). The circumferences of the three largest boli in each dung pile were measured to provide an estimation of elephant age (Tyson et al., 2002). All samples were stored at −20°C prior to export to the Eggert lab at the University of Missouri for genetic analyses.
Samples from the 2018/19 survey were compared with those from the 2006 genetic CMR survey of the Nakai Plateau (Ahlering, Hedges, et al., 2011) and the 2011 genetic CMR survey of the Sepon mine/Phou Xang He NPA (PXH NPA) area (Eggert & Ruiz-Lopez, 2012). Although the sequencing platform did not change between surveys, methods of amplification (singleplex vs. multiplex) and potential differences between allele calling by researchers make it difficult to compare studies (Ellis et al., 2011). Based on a 260/280 ratio closest to 1.80 with a DNA concentration greater than 15 ng/μL, the 10 highest quality samples were selected for amplification. DNA was extracted from the samples from Nakai 2018/19, Elephant Village, and 10 samples from Sepon 2011 using a modified QIAamp DNA stool minikit protocol (Archie et al., 2008) that was optimized by Budd et al. (2021).
Permissions for sample collection were obtained from the Government of Lao PDR, the NNT NP, and the NT2 Power Company (NTPC). This study was conducted in compliance with Article 17, paragraph 2, of the Nagoya Protocol on Access and Benefit Sharing (Certificate #ABSCH-IRCC-LA-256921-1).

| Genetic analyses of Nakai 2018/2019 samples
Samples were amplified at 18 multiplexed microsatellite loci (Eggert et al., 2000Kongrit et al., 2008; Table 1). Amplification products were visualized in an agarose gel, purified, submitted for fragment analysis in an ABI 3730xl DNA analyzer (Thermo Fisher Scientific) with added 600 LIZ size standard at the University of Missouri DNA Core Facility and scored following Budd et al. (2021) in GeneMarker v.1.9.7 (Soft Genetics). To determine the sex of individuals, samples were amplified at two short Y-specific fragments (SRY1 and AMELY2) and a longer X-specific fragment (PLP1), following Ahlering, Hailer, et al. (2011).
To quantify the minimum number of matching loci needed to identify individuals, genotypes were analyzed for probability of identity  pID < .001; pSibs < .01). We determined unique genotypes and recaptures in Cervus v.3.0.7 (Kalinowski et al., 2007) using the "fuzzy matching" function to identify genotypes that matched at all but three alleles.
Per locus diversity was estimated as observed heterozygosity (H O ), expected heterozygosity (H E ), Shannon's information index (I), and the mean fixation index/inbreeding coefficient (F IS ) in GenAlEx v.6.41, and allelic richness (A R ) and private allelic richness (A P ) in HP-Rare v.
1.1 (Kalinowski, 2005). We tested for relatedness between all females and young (dung bolus circumference < 30 cm) and all adult and subadult males were detected near each other within a 48 h period using Goodnight and Queller (1999) in GenAlEx v.6.41.
We tested for genetic differentiation between seasons using F ST and assessed significance levels using 9999 permutations in GenAlEx v.6.41. For individuals with a pairwise relationship of r > .25, one individual from the pairing was removed prior to the inference of genetic population structure using Structure v.2.3.4 (Pritchard et al., 2000) for up to 10 clusters (K) with 10 replicates for each K. We ran 50,000 burn-in steps and 250,000 MCMC replicates using an admixture model with no priors. We determined the best-supported number of genetic clusters using the ∆K method of Evanno et al. (2005) in Structure Harvester v.0.6.94 (Earl & VonHoldt, 2012).
For each unique genotype, we amplified an approximately 630 bp fragment of mitochondrial DNA (mtDNA) containing a portion of the C terminal of cytochrome b, the threonine and proline tRNAs, and the 5′ end of the noncoding control region (d-loop), using primers MDL3 and MDL5 (Fernando et al., 2000) and the conditions outlined in Eggert et al. (2002). We sequenced amplification products in both directions in an ABI 3730xl DNA Analyzer (Thermo Fisher Scientific) and aligned and edited sequences in Geneious v. 8.0.5 (Kearse et al., 2012).
Haplotypes were collapsed using FaBox v.1.41 (Villesen, 2007) and compared to those in GenBank for sequence similarity. Team, 2018). Significant differences in diversity between populations were determined using general linear models with locus as a fixed effect using the Lme4 package v.1.1-23 (Bates et al., 2015) with A R and A P transformed to gamma distributions. These tests were followed by an ANOVA in the package car v.3.0-9 (Fox & Weisberg, 2019) and post-hoc testing using Tukey tests in multcomp v.1.4-13 (Hothorn et al., 2008).

| Genetic and demographic comparisons of Nakai 2018/2019, Nakai 2006, and Sepon 2011
Comparison of the proportion of the total genetic variance contained within each population relative to the total genetic variance (F ST ) was performed in GenAlEx v.6.41 using 9999 permutations with a standard Bonferroni correction to determine statistical significance (Neyman & Pearson, 1928 Excoffier & Lischer, 2010), assessing the significance of differentiation using 1000 permutations and applying a standard Bonferroni correction (Neyman & Pearson, 1928).
We assessed demographic differences between Nakai 2018 (dry season), Nakai 2019 (wet season), and Nakai 2006 (dry season) using fecal bolus circumferences as a proxy for elephant age (Tyson et al., 2002). We tested for deviations from the normality of distri-   Table 2).
HEC caused by elephants likely dispersing from the Nakai Plateau also occurred across the wider landscape ( Figure 1c). During fieldwork by WCS staff in and around the Sepon mine area, some 100 km from the Nakai Plateau, monitoring teams were told by farmers that HEC had increased over the previous years.
Monitoring of the G3 elephants indicated that they had ex-

| Comparisons between the Nakai studies of 2006, 2018, and 2019
We successfully re-genotyped 10 individuals from Nakai 2006  We identified 64 individuals that had at least one relative at the level of r > .25, with 31 having at least 1 relative at the r ≥ .50.
From these individuals, we identified 13 relative groups united by probable parent-offspring, full-sibling, or half-sibling relationships.
Nine of these relative groups consisted of only two full-sibling in-  (Table 4).

TA B L E 4
Genetic differentiation based on the nine nuclear microsatellite loci shared among studies (F ST ) below diagonal and mtDNA (ɸ ST ) above diagonal; significance following Bonferroni correction is indicated by *. coincided with the inundation of the Nakai Plateau in April 2008 (Hedges & Hallam, 2011).
The G3 elephants became a particular problem, as they displayed little fear of humans, had become habituated to living in areas with villages and agriculture (Tyson & Rasphone, 2013), and were involved in at least one human death (Tyson & Phakphothong, 2015).
This contrasts with the PXH NPA elephants living adjacent to the Sepon mine area which also caused HEC, but avoided contact with people, based on radio-collaring data (NTPC, unpublished), despite their very close proximity. Because attempts in January 2018 and November/December 2019 to radio-collar at least one of the G3 elephants (Tyson & Stremme, 2020) were unsuccessful, the ability to detect their movements and activities continues to be limited.
Since  We also detected a shift in the age structure of the population to a higher proportion of sub-adults. In 2018 and 2019, populations were comprised of a relatively large number of subadults representing 53% and 42% of the population in the 2018 dry season and the 2019 wet season, respectively. In the 2006 dry season survey, subadults accounted for only 31% of the population.
The loss of nuclear genetic diversity following the construction of the NT2 dam is concerning, as the loss of diversity can affect long-term population viability (Reed & Frankham, 2003 (Kongrit et al., 2008), Cambodia (Gray et al., 2014), and Myanmar (Kusza et al., 2018).
In addition, the significantly lower allelic richness of 2019 compared to 2006 and 2018 was likely due to the number of close relatives found in the detected genotypes. We found a single male with 17 close relatives (.125 ≥ r < .50). Although this male likely only fathered three of those relatives, such high reproductive output by a few individuals in a small population can result in a rapid decline in effective population size (Caballero, 1994).
Having committed to a 23% increase in renewable energy by 2025 (ASEAN Centre for Energy, 2021), the countries of Southeast Asia face the challenge of developing the infrastructure needed.
This will inevitably entail significant changes in land use to accommodate solar, wind, and hydropower. In their analysis of the potential for energy development in the region, Sakti et al. (2023) found that areas in northern Southeast Asia (Vietnam, Laos, Thailand, and the Philippines) had the highest potential for developing power from all three sources. The intention of Lao PDR to become the "battery of Southeast Asia" has resulted in over 50 hydroelectric dams in 15 years, with a further 101 under construction or planned (Chang, 2013;Williams, 2019). A study of existing and planned dams worldwide found that 1249 large dams are located in protected areas and that 509 new dams are currently planned (Thieme et al., 2020).
While dams may benefit some species through, for instance, the creation of artificial wetlands, protected areas that contain dams cannot be considered to offer protection to the associated ecosystems (Sakti et al., 2023).
This study provides a greater understanding of not only the direct local impacts of hydropower projects on elephant populations, but also the landscape-level effects of such projects, which to date have remained under-appreciated and not included in mitigation plans. Future development of infrastructure projects should take into account potential landscape-level (i.e., distant) impacts of altering key habitat for wide-ranging species such as elephants, and should include assessments of potential human-wildlife conflict, including the economic impacts, and identify appropriate mitigation measures. The recent development of models that jointly consider habitat connectivity and human-wildlife conflicts and guidelines on human-wildlife conflict and coexistence (IUCN, 2023;Vasudev et al., 2023) are promising as they may provide a framework for conservation planning.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare that they have no conflicts of interest with respect to this work.

DATA AVA I L A B I L I T Y S TAT E M E N T
Microsatellite genotype data has been deposited in DRYAD https://doi.org/10.5061/dryad.dv41n s247 and mitochondrial DNA sequence data is available on GenBank under the accession numbers provided.